The invention relates to the area of mass spectrometry and, more particularly, is concerned with a method of high-throughput, comprehensive tandem mass spectrometry in apparatus, including two time-of-flight mass spectrometers.
Mass spectrometers are devices which vaporize and ionize a sample and then use static or dynamic electric fields to measure the mass-to-charge ratios of the ions formed. Tandem mass spectrometry is used for structural analysis and the identification of compounds in complex mixtures. In every application the MS-MS procedure has the same sequence of operations:                mass selection of parent ions of a single mass-to-charge ratio (m/z);        fragmentation of those ions; and mass        analysis of the fragments.Although there is a large variety of tandem MS-MS instruments with their own strength and weakness, all of them have one common feature—all of them use one parent ion at a time. The rest of ion species are removed out of the primary ion beam and lost.        
Triple quadrupole instruments are the most common MS-MS instrument. A continuous ion source, e.g., electrospray (ESI), introduces ions into a first quadrupole mass filter, which is tuned, such that only ions of interest pass the mass filter. The rest of the primary beam components are rejected and lost. Selected ions are transmitted into a so-called “collision induced dissociation” (CID) cell, filled with gas at mtorr pressures and equipped with a radio frequency (RF) quadrupole guide. The kinetic energy of the injected ions is controlled by an electrostatic bias on the mass filter and is adjusted to induce ion fragmentation via gas collisions. Fragment ions are collisional dampened in a CID cell and then introduced into a second quadrupole for mass analysis. Since mass scanning in a second quadrupole takes time and causes additional ion losses by factor of c.a. 1000, triple quadrupole instruments are mostly used for detection of known species with known masses of parent and fragment ions.
The introduction of quadrupole time-of-flight tandem mass spectrometers (Q-TOF) strongly enhanced throughput of MS-MS instruments (see Morris et al., Rap. Comm. Mass. Spectrom., v. 10, pp. 889–896, 1996). The triple quadrupole was modified, such that the second quadrupole mass filter was replaced by an orthogonal TOF MS (oa-TOFMS). This substitution gave an advantage of parallel analysis of all fragment ions at once and, hence, higher sensitivity and faster acquisition in a second MS, as well as enhanced resolution and mass accuracy of a second MS. However, the quadrupole is still used for parent ion selection, accompanied by rejection of all ion species but one. The idea of parallel analysis has not been extended parent ions.
Another common MS—MS device uses Paul ion trap mass spectrometer (ITMS), well described in March, R. E., Hughes R, J. Quadrupole storage mass spectrometry, Willey-Interscience, New York 1989. Ions, produced in the ion source, are periodically injected into an ITMS and are trapped within the ITMS by a radio frequency (RF) field. “Unwanted” species are removed, e.g., by applying a broadband resonant AC signal, so that only ions of interest remain in the trap. Selected parent ions are then excited by a separate AC field, resonant with the secular motion of the precursor. Parent ions gain kinetic energy and fragment in energetic collisions with a buffer gas. Fragments are mass analyzed using a resonant ejection technique. The amplitude of an RF field is ramped such that ions leave the trap sequentially according to their m/z values.
It also has been known to couple a 3-D Paul trap with a TOF analyzer for more accurate mass analysis of fragment ions. See Quin and D. Lubman, Rap. Comm. Mass. Spectrom., 10, 1079, 1996 and WO 99/39368 by Shimadzu. A linear ion trap (LIT) has been coupled to a TOF analyzer in U.S. Pat. No. 5,847,386 by Thomson et al., U.S. Pat. No. 6,111,250 by B.A. Thomson and L.L. Joliffe, U.S. Pat. No. 6,020,586 by T. Dresch et al. and WO 01/15201 by B. Reinhold and A. Verentchikov. All ion trap tandems are mostly oriented on multiple stage MS—MS analysis. Parent ions are selected with a loss of other ion components.
Recently introduced tandem time-of-flight mass spectrometers (TOF—TOF) are the closest prototypes to the below described invention by similarity of employed hardware. Examples of TOF—TOF are described in U.S. Pat. No. 5,032,722 by Schlag et al., U.S. Pat. No. 5,464,985 by T. J. Kornish et al., U.S. Pat. No. 5,854,485 by T. Bergmann, WO 99/40610 by M. L. Vestal, and WO 99/01889 by C. Hop. In all TOF—TOF tandems, a pulsed ion beam is time separated in a first, high-energy TOF and filtered by timed ion selector, so that only ions of interest pass into the CID cell. The CID cell is filled with gas at a low gas pressure (usually below 1 mtorr) to induce single high energy collisions with the buffer gas sufficient for ion fragmentation, but still retaining short duration to maintain an ion packet. A pulsed beam of fragment ions is analyzed in a second, high energy TOF. To handle the large energy spread of the fragment ions, the second TOF employs either quadratic field potential or an additional pulsed acceleration.
In WO 00/77823 by A. Verentchikov, a variation of TOF—TOF tandem employs slow injection of parent ions into a CID cell with collisional dampening of fragments and subsequent injection into an orthogonal TOF. The instrument is the closest prototype of the invention, considering employed components. Collisional dampening in the fragmentation cell improves ion beam characteristics upstream of the second TOF and allows high resolution and accurate measurements of fragment ion masses. The first TOF operates at 1 kV energy and a short time scale. A time gate in front of a CID cell admits only one parent ion mass at a time.
In all described tandems the first mass analyzer (either quadrupole, ion trap or TOF) selects one parent ion mass at a time and rejects all other components. In some applications, like drug metabolism studies, it is acceptable to follow a single compound of interest. In the case of complex mixtures (like protein characterization out of gels), however, it is necessary to analyze multiple parent ions. Using existing techniques, sequential MS—MS analysis of multiple precursors is tedious and insensitive.
Recently introduced tandem IMS-CID-TOF mass spectrometers employ a principle of time-nested acquisition, potentially to be implemented without ion losses. See WO 00/70335 by D. Clemmer. Since separation in the ion mobility spectrometer (IMS) occurs in milliseconds and TOF mass spectrometry in microseconds, it is possible to acquire fragment spectra for each ion mobility fraction. The disadvantage of the technique is a poor IMS separation with mobility resolution below R=50, which corresponds to mass resolution of about 10. Since IMS-TOF tandem employs a principle of comprehensive tandem mass spectrometry with time-nested acquisition, it is selected as a prototype of the invention.
The idea of MS—MS analysis without parent ion losses is also disclosed in WO 01/15201 by B. Reinhold and A. Verentchikov. Ions are selected by resonant excitation and moved between ion traps without rejecting other ionic components. The procedure is tedious and long, while ions from the ion source are lost. So-called parallel ion processing is employed in multiple ion traps in WO 92/14259 by Kirchner, where the beam is split between multiple traps. Time is saved by sacrificing sensitivity.
There is still a need for an instrument providing rapid and sensitive MS—MS analysis for multiple parent ions in parallel without rejecting ions coming from an ion source. Such an instrument would further improve a throughput of MS—MS analysis, desirable in analysis of complex mixtures.